Tag: horticulture

In order to elucidate the biochemical mechanism of BZS1 function, we performed a SILIAIP-MS analysis of the BZS1 protein complex. We transformed Arabidopsis with a construct that over expresses a BZS1 protein fused with the yellow fluorescence protein at the C-terminus driven by the constitutive 35S promoter . A transgenic line that showed mild dwarf and dark-green-leaf phenotypes, resembling the bzs1-D mutant , was selected for the analysis. Pair-wised comparison was designed to seperately compare BZS1-YFP and 35S::YFP transgenic plants with non-transgenic wild type, to determine proteins associated with BZS1-YFP and YFP alone, respectively. To obtain complete 15nitrogen labeling of young seedlings, we first grew BZS1-YFP, YFP and wild-type plants hydroponically in medium containing 15N, and obtained stable isotope-labeled seeds . These 15N-labeled seeds and regular 14N seeds were grown again on corresponding 15N or 14N medium to obtain 5-day-old seedlings for further analysis . For each pair of isotope-labeled sample and control, equal amount of tissues was mixed, and the protein extract was used for immuno precipitation using the GFPtrap beads. The immuno precipitated proteins were separated in SDS-PAGE, gel bands were in-gel digested, and the tryptic peptides were analyzed by mass spectrometry . Mass spectrometry analyses of the two BZS1-YFP immuno precipitation experiments identified 514 and 383 proteins, respectively, with 279 proteins identified in both repeats . A smaller number of proteins were identified in the YFP experiments . Quantitation of isotope ratios showed median ratios of 1.16 and 1.23 for the two BZS1-YFP experiments,planting gutter and 1.0 and 0.92 for the two YFP control experiments. The protein ratios of the YFP control datasets had standard deviation of 0.23 and 0.57 .

Using 2× median as cutoff, 16 proteins were enriched in BZS1-YFP compared to wild-type control in the two repeat experiments. The YFP and wild type comparison identified 2 proteins that were enriched over 2× median, presumably due to association with YFP or false discovery, suggesting a false discovery rate <0.8% . The 15 proteins enriched by BZS1-YFP were not enriched by YFP alone, and thus were considered BZS1-associated proteins . Among the BZS1-associated proteins are COP1 and HY5, two key regulators of the light signaling pathways, as well as BZS1/BBX20’s homologs STH2/BBX21 and STO/BBX24 . To verify the interaction between BZS1 and COP1 in vivo, we performed immuno precipitation of BZS1-YFP from the BZS1-YFP transgenic Arabidopsis seedlings using anti-GFP antibody, and probed the immunoblot with anti-COP1 antibody. The results showed that COP1 co-immuno precipitates with BZS1-YFP , confirming that BZS1 interacts with COP1 in plants. Consistent with BZS1’s interaction with the COP1 E3 ubiquitin ligase, the immuno precipitated BZS1-YFP can be detected by anti-ubiquitin antibody, and the level of ubiquitination was increased by treatment with proteasome inhibitor MG132 . We further confirmed the direct interaction of BZS1 and HY5 by yeast two-hybrid assays . Further, when transiently co-expressed in Nicotiana benthamiana, the BZS1-myc protein was co-immuno precipitated by the HY5-YFP protein , confirming their interaction in plant cells. Similarly, the STH2-myc protein was co-immuno precipitated by BZS1-YFP . These results confirmed the SILIA-IP-MS results that BZS1 interacts with COP1, HY5, and STH2/BBX21. To determine the functional relationship between BZS1 and HY5, we first compared previously published transcriptomic data from BZS1-overexpression plants with chromatin immuno precipitation-microarray data of HY5 direct target genes . The result showed that 56.3% of BZS1-activated genes are HY5 targets while only 13% of BZS1-repressed genes are HY5 targets . Such significant overlap betweenBZS1-activated and HY5-bound genes suggests that BZS1 interacts with HY5 to activate gene expression.

Fusing a transcription repressor domain, such as the SRDX domain, to a transcription activator has been shown to have a dominant negative effect . Over expression of the BZS1-SRDX fusion sequence driven by 35S promotor in Arabidopsis caused a long-hypocotyl phenotype and reduced anthocyanin accumulation , which were similar to the phenotypes of loss-of-function mutant hy5-215 but opposite to the phenotypes caused by BZS1 over expression, further supporting that BZS1 functions as a transcription activator together with HY5. The BZS1-SRDX plants grown in the dark did not show any obvious phenotype , consistent with HY5 and BZS1 being degraded in the dark. To further investigate whether BZS1 function requires HY5, we crossed BZS1-YFP with hy5-215. The BZS1-YFP/hy5-215 plants showed similar phenotypes of long hypocotyls and low anthocyanin accumulation as hy5-215 , demonstrating that BZS1 activity requires HY5. Interestingly, the BZS1-YFP protein accumulates at a higher level in the hy5-215 mutant than in wild-type background , suggesting that HY5 negatively regulates BZS1 accumulation while required for BZS1 function. On the other hand, the RNA levels of HY5 and HYH are higher in BZS1-YFP line but lower in BZS1-SRDX seedlings as compared with those in wild type . Immunoblot analysis also confirmed that the HY5 protein level was increased in the BZS1-YFP line and reduced in the BZS1-SRDX line . These results indicated that BZS1 and HY5 proteins not only interact directly, but also influence each other’s protein abundance. A previous study showed that HY5 is required for SL inhibition of hypocotyl elongation. The HY5 protein level is increased by SL treatment and the hypocotyl elongation of hy5 is partially insensitive to SL . Since BZS1’s function is dependent on HY5 in the light, we examined if BZS1 is also involved in SL signaling. As reported previously , treatment with 1 μM GR24, an analog of SL, dramatically inhibited the hypocotyl elongation of wild-type seedlings but had no effect on the SL insensitive mutant max2-3 . We found that the hypocotyl elongation of BZS1-SRDX seedlings was partially insensitive to GR24, similar to the hy5-215 mutant.

The GR24 treatment decreased the hypocotyl length of wild-type seedlings by about 72% compared to the untreated control, but only by about 17% for hy5-215 and 30% for the BZS1-SRDX seedlings . GR24 also increased the chlorophyll content in wild-type plants by about 24%, but had no significant effect in max2-3, hy5-215 and BZS1- SRDX seedlings . Additionally, GR24 induced HY5 accumulation in wild-type background but not in the BZS1-SRDX seedlings . These results indicated that, like HY5, BZS1 also plays an important role in SL regulation of hypocotyl elongation and chlorophyll accumulation. We then tested if SL regulates the expression of BZS1/BBX20 and its homologs. Real-time reverse transcription PCR analysis showed that GR24 increased the expression level of BZS1/BBX20 mRNA in wild type, but not in the max2-3 mutant . Interestingly, expression levels of other members of BBX IV family, including STH2/ BBX21, were not dramatically affected by GR24. Immunoblot analysis confirmed that GR24 treatment increased the levels of the BZS1-myc protein expressed from the BZS1 native promoter and the BZS1-YFP protein expressed from the constitutive 35S promoter, suggesting that SL regulates BZS1 at both transcriptional and post transcriptional levels . These results indicated that BZS1 plays a positive role in SL signaling downstream of MAX2 at the early stage of seedling development. Seedling development is crucial for establishment of life for a plant, and is thus highly responsive to a wide range of environmental and hormonal signals. The signaling pathways that transduce these signals are highly integrated at the molecular level to ensure coherent cellular responses and optimal growth according to environmental condition and endogenous physiology . This study uncovers additional mechanisms for such signal integration. Our quantitative proteomic analysis of the BZS1 complex reveals BZS1’s interaction with HY5,gutter berries as well as provides direct evidence for in planta BZS1-COP1 interaction. Genetic analyses using over expression and dominant negative loss-of-function transgenic plants demonstrate that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. Further, we find that BZS1 also mediates SL regulation of HY5 level and hypocotyl elongation. Together with previous finding of BZS1 function downstream of the BR pathway , our study establishes BZS1 as a key integrator of light, BR, and SL signals for regulating seedling morphogenesis. IP-MS is a powerful method for identification of interacting proteins, which has been widely used in dissecting signal transduction pathways . With increased sensitivity of modern mass spectrometers, IP-MS tends to identify not only specific interacting proteins but also large numbers of non-specific proteins. Under our experimental conditions, over 300 proteins were identified in each IP-MS analysis. Distinguishing specific from non-specific interactors is challenging without quantitative measurement. SILIA-IP-MS provides an ideal quantitative method for this purpose, as the sample and negative control can be mixed at an early step of the immuno precipitation experiment to avoid technical variations. Indeed, among the large numbers of proteins identified by mass spectrometry, only 29 showed enrichment by the BZS1-YFP fusion protein, and thus were considered BZS1-associated proteins. The interactions of BZS1 with HY5, COP1, and its homolog STH2/BBX21 were confirmed by yeast two-hybrid or coimmuno precipitation assays. Consistent with COP1-mediated ubiquitination of BZS1, our BZS1-interactome data includes ubiquitin and one proteasome activating protein PA200 .

In theory, the ratio between sample and negative control should be infinite for proteins that specifically interact with the bait protein in SILIA-IP-MS. However, due to background signals in the control samples, either from non-specific binding of proteins in immuno precipitation or interfering signals in MS1, the ratios actually distribute within a wide range. For example, Hubner et al. observed that pull-down with Aly-GFP leads to only moderate enrichment because Aly itself binds to control beads as well. In our study, only 2 of the 254 proteins identified in the YFP sample were enriched over 2× median, suggesting that even 2-fold cutoff yields low false discovery rate when two reverse-labeled replicates are used. Our genetic analyses support that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. First, comparison of genome-wide data shows that BZS1 tends to activate, rather than repress, HY5 direct target genes . Second, dominant inactivation of BZS1 causes similar phenotypes as the hy5-215 mutant , supporting that BZS1 and HY5 act in the same or overlapping pathway. Third, the phenotypes of BZS1-YFP plants are suppressed by hy5-215 , confirming that BZS1 functions in a HY5-dependent manner. These results together provide strong evidence for a model that BZS1 interacts with HY5 to activate HY5-bound target genes. BBX proteins contain one or two B-box zinc finger motifs in their N-terminal regions, and are organized into five subfamilies . The fourth subfamily includes eight B-box proteins containing two tandem B-boxes without CCT domain . Our study together with previous studies show that five members of the BBX subfamily IV interact with COP1 and HY5 . Thus, interaction with HY5 seems to be a common mechanism for these B-box proteins to regulate gene expression. Interestingly, BZS1/BBX20, STH2/BBX21 and LZF1/STH3/BBX22 are positive regulators in photomorphogenesis, while BBX19, STO/BBX24 and STH/BBX25 are negative regulators . Our finding of STH2/BBX21 and STO/BBX24 as interactors of BZS1/BBX20 suggests that these factors form hetero-dimers. The dominant negative effect of the BZS1-SRDX fusion indicates that BZS1/BBX20 normally functions as a transcription activator, which is consistent with previous finding that STH2/BBX21 functions as a transcription activator . It has been reported that STO/BBX24 and STH/BBX25 interact with HY5 and most likely inhibit HY5 function by forming inactive heterodimers . Our identification of STO/BBX24 as a BZS1-associated protein suggests another possibility that STO/BBX24 may form a non-functional heterodimer with BZS1/BBX20 and hence inhibit BZS1/BBX20 activity. In addition to direct interaction between BZS1 and HY5 proteins in regulating target gene expression, BZS1 and HY5 also regulate each other’s expression level. BZS1 positively regulates the RNA and protein levels of HY5 . Recent studies have shown that HY5 binds to its own promoter to regulate its own level , thus BZS1 may regulate HY5 transcription through interaction with HY5 protein. In contrast, the BZS1 protein level is increased in hy5-215, suggesting a negative regulation by HY5 at the protein level. HY5 may promote BZS1 degradation by interacting with COP1. Similarly, a previous study showed that the degradation of BBX22 is also promoted by both COP1 and HY5 , whereas BBX22 transcription is directly activated by HY5 and repressed by BBX24 . Such positive and negative regulation between interacting partners potentially contributes to the signaling dynamics during dark-to-light transition and fluctuating light intensities.Our study uncovers a major role for BZS1 in SL response.

As sessile organisms, plants are presented with numerous biotic challenges such as herbivory and pathogen attack. Plants initiate responses to these challenges by harnessing tightly regulated phytohormone networks. Salicylic acid levels increase in plants following pathogen infection and SA is critical for the development of systemic acquired resistance . There are two enzymatic pathways for the generation of SA: one via phenylalanine ammonia lyase and the other via isochorismate synthase . In tomato , Arabidopsis and Nicotiana benthamiana, most pathogen-induced SA appears to be synthesized via the ICS pathway . Plants with compromised SA synthesis or signaling have greatly diminished defenses against pathogens, as is the case with SA-deficient transgenic plants expressing a bacterial salicylate hydroxylase or ICS mutants like sid2 , and mutants in downstream targets of SA such as npr1 . SAR induction by biotic agents coincides with increases in SA levels and a systemic transcriptional reprograming that primes the plant to respond rapidly to minimize the spread or severity of further infections . This transcriptional reprograming includes the expression of pathogenesis-related genes and deployment of peroxidases and other defense factors. In addition to induction by biotic agents, SAR responses are induced by exogenous application of SA to the foliage or roots . Plant activators are chemicals that have no direct antimicrobial activity but induce disease resistance . A number of synthetic compounds have been developed that induce SAR by increasing SA accumulation and/or by acting on downstream targets of SA . For example, the plant activator, probenazole, effective against bacterial, fungal, and oomycete diseases, stimulates SAR by increasing SA levels . 1,2,3-Benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester , sold under the trade name, Actigard,grow bucket stimulates SAR in many plant species without inducing SA accumulation . Tiadinil [TDL; N–4-methyl-1,2,3-thiadiazole-5-carboxamide] is a plant activator that was registered in Japan in 2003 under the trade name, V-GET. TDL was developed for disease management in rice where it is applied to nursery-grown seedlings for transplanting to production fields . TDL is very effective for control of rice blast disease caused by Magnaporthe oryzae and appears to induce resistance in a manner similar to BTH by acting on downstream targets of SA .

The TDL metabolite,4-methyl-1,2,3-thiadiazole-5-carboxylic acid, is responsible for the SAR activation .The effect of brief episodes of root stress such as salinity and water deficit at levels that commonly occur in agriculture is well documented in plant–oomycete interactions, wherein stress events predispose plants to levels of inoculum they would normally resist . The phytohormone abscisic acid accumulates rapidly in roots and shoots as an adaptive response to these abiotic stresses, but also contributes to the increased disease proneness of the plants . Antagonism between SA and ABA is well documented in relation to plant defense responses to pathogens . Previously, ABA was found to have an antagonistic effect on SAR which was induced by 1,2-benzisothiazol-3-one1,1-dioxide and BTH in Arabidopsis and tobacco . However, it is not known if plant activators that target SA signaling impact the ABA-mediated susceptibility to root pathogens that occurs following predisposing root stress in tomato. Because of the potential for unwanted trade offs and signaling conflicts in plants exposed to different stresses, as can occur in the field, we investigated how predisposing root stress impacts chemically induced resistance in tomato. The objective of this study was to determine the effect of pretreatment of tomato seedlings with TDL and BTH on salt-induced predisposition to the foliar bacterial pathogen Pseudomonas syringae pv. tomato and to the soil borne oomycete pathogen Phytophthora capsici. TDL is of particular interest in the context of soil borne pathogens such as Phytophthora capsici because it is often applied to plants as a root dip. We also determined the impact of SA, TDL and BTH on ABA accumulation during a predisposing episode of salt stress. The results show that TDL applied to roots strongly protects the leaves from disease caused by Pst in both non-stressed and salt-stressed plants. In contrast, neither TDL nor BTH protects roots from Phytophthora capsici.

The protection induced by plant activators against Pst does not result from reduced ABA accumulation and, although overall disease is less in both non-stressed and salt-stressed plants by chemically induced SAR, plant activators do not reverse the salt-induced increment in disease severity.To determine the effect of SA on ABA accumulation during salt stress, ABA levels were measured in WT plants pre-treated with SA, TDL, or BTH. Following salt stress treatment for 18 h, roots and shoots were collected and immediately frozen in liquid N2.The tissues were lyophilized and placed at −20◦C until extraction. The lyophilized tissue was ground in liquid N2 to a fine powder with a mortar and pestle, 50–100 mg samples were collected, and each sample transferred to a micro-fuge tube. Cold 80% methanol containing butylated hydroxytoluene at 10 μg ml−1 was added to each tube, which was then vortexed. The extracts were placed on ice and agitated occasionally for 30 min. The tubes were centrifuged for 5 min at 10,000 × g, and the supernatants collected. The pellet was extracted with 0.5 ml of 80% methanol and centrifuged to collect the supernatant. This step was repeated, all three supernatants were combined, and the methanol concentration of the extract adjusted to 70%. The extracts were applied to pre-wetted Sep-pak C18 columns and eluted with 5 ml of 70% methanol. The eluate containing ABA was concentrated to near dryness at 37◦C under vacuum and the volume adjusted to 300 μl with deionized water. The samples were analyzed by competitive immuno assay with an ABA immuno assay kit according to the manufacturer’s directions. Results are expressed as nanomoles of -ABA per gram dry weight of tissue. To determine the effect of the nahG transgene on ABA levels, roots and shoots from WT and NahG plants were processed using the same procedure as above.To determine if plant activators induce resistance to Pst under different stress regimes in our experimental format, roots of hydroponically grown seedlings of cv. “New Yorker” were treated with TDL and then either not salt-stressed or exposed to 0.2 M NaCl for 18 h prior to inoculation.

In preliminary experiments, several concentrations of TDL were evaluated for phytotoxicity and for efficacy against bacterial speck disease with 10 ppm TDL selected as this concentration provided an optimal response. Concentrations higher than 10 ppm of TDL caused a slight bronzing of the roots and depressed growth of the seedlings, suggesting a mild phytotoxicity of the chemical in our experimental format at these higher levels. Inoculated salt-stressed seedlings had more severe disease symptoms and a significantly higher titer of pathogen than non-stressed, inoculated plants. Pretreatment with TDL at 10 ppm significantly reduced Pst colonization and symptom severity in “New Yorker” plants in both non-stressed and salt-treated seedlings . However, TDL did not prevent the proportional increase in Pst colonization observed in salt-stressed plants relative to the non-stressed controls.Since TDL harnesses SA-mediated defenses, we treated SA deficient NahG plants to see if TDL induces resistance under the different stress regimes in this highly susceptible background. As expected, NahG plants were more susceptible to Pst and accumulated significantly less SA following Pst infection than the WT background “New Yorker.” However, TDL provided strong protection in the NahG plants and mitigated the predisposing effect of salt-stress on bacterial speck disease.In a previous study we showed that ABA-deficient tomato mutants displayed a much reduced predisposition phenotype to salt stress . To determine if the protective effect of TDL is altered within an ABA-deficient tomato mutant,dutch bucket for tomatoes seedlings of WT and an ABA-deficient mutant within this background, sitiens, were treated in the same format and stress regimes as above. TDL significantly reduced Pst symptoms and colonization in both non-stressed and salt-treated plants of “Rheinlands Ruhm.” However, 3.6- and 5.4-fold increases in pathogen titer as a result of salt-stress were observed in both the control and TDL-treated plants, respectively, indicating that TDL did not prevent the proportional increase in Pst colonization in salt-stressed plants, similar to the results with “New Yorker” and NahG plants. In contrast, the sitiens mutant was not predisposed to Pst by salt stress and had significantly reduced symptoms and colonization by the pathogen than the background “Rheinlands Ruhm” . Nonetheless, TDL pretreatment of sitiens provided further protection against Pst .To determine if plant activators protect tomato roots and crowns against the oomycete pathogen, Phytophthora capsici, and predisposing root stress, tomato seedlings were treated with TDL or BTH , not stressed or salt-stressed as above, and then inoculated. There was no protection provided by the plant activators against disease caused by Phytophthora capsici in either the control or salt-treated plants, as reflected in symptom severity and pathogen colonization .Because elevated levels of ABA in tomato can enhance susceptibility to Pst and Phytophthora capsici, the effect of SA, TDL, and BTH on ABA levels was determined in roots and shoots. ABA concentrations in either shoots or roots at the time selected for inoculation in our treatment sequence were not altered by SA . However, a trend of increasing ABA accumulation was observed in TDL- and BTH treated “New Yorker” plants relative to the corresponding control plants . Although the increase in ABA accumulation in the plants treated with these plant activators is not statistically significant at P ≤ 0.05, it can be said that SA, TDL, and BTH do not reduce ABA content relative to untreated plants . In addition, salt stress did not further increase the levels of ABA in plants that had been pretreated with TDL or BTH, which were similar to the salt stressed controls.In a previous study, we demonstrated the predisposing effect of salt stress and a role for ABA as a determinative factor in predisposition in the tomato–Phytophthora capsici interaction .

The present study is the first report of salt-induced predisposition to the bacterial speck pathogen, Pst, in tomato. Furthermore, the results with the ABA-deficient sitiens mutant are consistent with the salt-induced susceptibility to Pst being mediated by ABA . These results conform to studies in Arabidopsis where ABA has been reported to promote susceptibility to Pst .Because SA has been shown to protect tomato against salt stress, possibly by an ABA-dependent mechanism , plant activators that operate via the SA pathway were evaluated for effect on salt-induced predisposition. Protection of tomato against bacterial speck disease by BTH is well documented , and TDL has previously been shown to reduce the severity of bacterial and fungal infections without inducing SA accumulation . Here, TDL was shown to protect against Pst in both non-stressed and salt-stressed tomato plants. TDL pretreatment strongly reduced disease and colonization by Pst in both “New Yorker” and SA-deficient NahG plants. TDL, or more likely its biologically active metabolite, SV-03, presumably allows the NahG plants to mount an SAR response to Pst infection in the absence of SA accumulation . TDL provided protection in both non-stressed and salt-stressed plants, but did not reverse the predisposing effect of salt stress. An increase in Pst colonization was observed in the salt-stressed, TDL-pretreated plants of both genotypes, with comparable percentage increases relative to the corresponding non-stressed controls in “New Yorker” and NahG plants. This indicates that TDL does not reverse the salt-stress effect on disease, per se, and likely targets stress network signaling independently of an ABA-mediated process that conditions the salt-induced susceptibility observed in this system . “Rheinlands Ruhm” also displayed salt-induced predisposition to Pst. Pretreatment with TDL significantly reduced Pst colonization in both “Rheinlands Ruhm” and sitiens . Similarly, TDL provided protection in both non-stressed and saltstressed plants, but did not reverse the predisposing effect of salt stress in “Rheinlands Ruhm” plants. The salt-induced increment in colonization by the pathogen was comparable in both the untreated and TDL-treated plants . The ABA-deficient mutant, sitiens, is considerably less susceptible to Pst than its background “Rheinlands Ruhm,” and does not exhibit salt-induced predisposition .Protection by plant activators against foliar pathogens is well established . However, relatively few studies have examined these compounds against soilborne pathogens and so TDL and BTH were evaluated for protection against root infection by Phytophthora capsici. Neither TDL nor BTH induced resistance or impacted salt-induced predisposition to Phytophthora capsici . Phytophthora capsici is an aggressive root and crown pathogen with a hemibiotrophic parasitic habit that triggers both SA- and jasmonic acid-mediated responses during infection of tomato .

The whole-plant N2 fixation potential was calculated by multiplying the total dry nodule biomass of each plant and the N2 fixation potential, which had been normalized to dry nodule biomass. To understand how plant effects were related to CNM concentration-dependent agglomeration in moist soils, the short- and long-term stabilities of CNMs were studied in soil water extracts. Briefly, control soil was weighed into separate 50 mL centrifuge tubes with 1:5 w/v Nanopure water . The centrifuge tubes were sealed securely and shaken horizontally on a shaker for 3 h . The extract was centrifuged to separate large solids, and the supernatant was decanted. The supernatant was vacuum filtered through a 0.22 μm membrane filter , and the filtrate was collected as the final soil extract and stored prior to use. A CNM stock solution was prepared by weighing dry CNM powder into the filtered soil extract, then mixing by brief sonication using a Branson 1510 bath sonicator . Aliquots of the dispersed CNM stock solution were further diluted by the filtered soil extract to yield a final lower concentration of 10 mg L−1. These two CNM concentrations were chosen for comparing the effect of lower versus higher CNM concentrations on CNM agglomeration in moist soil; both concentrations are relevant to the CNM doses used in the plant exposure experiment . The CNM suspensions were bathsonicated immediately before use in static agglomeration and sedimentation studies, hydroponic nft channel which were performed over a long time period . The changes of CNM hydrodynamic diameter and derived count rate with time were measured using dynamic light scattering in a Zetasizer NanoZS90 . DLS measurements were made every 15 s for the first 12 h, then daily from 1 to 7 d, and finally weekly until 56 d.

Meanwhile, dynamic CNM sedimentation in the soil extract was monitored by measuring the suspension absorbance at 600 nm using a UV-1800 spectrophotometer . Sedimentation patterns were inferred from the time course of normalized suspension absorbance at 600 nm . The UV-1800 spectrophotometer was zeroed using Nanopure water. The absorbance of the filtered soil extract alone was monitored over time as well, to confirm there was no interfering absorbance from the soil extract in the CNM suspensions. The zeta potential and electrophoretic mobility of the filtered soil extract and of 10 mg L−1 CNMs were also obtained using the Zetasizer NanoZS90. For either DLS, absorbance, ζ potential, or EPM, at least three replicate measurements were performed. Environmental scanning electron microscopy was performed to visualize the agglomerate morphologies of 10 and 300 mg L−1 CNMs in the soil extract, against a clean quartz sand substrate. Specimens were prepared by dispensing approximately 100 μL of the CNM suspensions onto clean quartz sand overlaying a 10 mm stainless steel conical-well Peltier stub. Imaging was by an FEI Co. XL30 field emission gun microscope , operated at 15 kV accelerating voltage, in a 3.5-torr chamber pressure with a gaseous secondary electron detector in environmental mode. Data are shown as the mean ± SE . For each CNM type, one-way analysis of variance with Tukey’s or Games-Howell post hoc multiple comparisons was used to determine significant differences between treatments . Homogeneity of variance was tested with Levene’s test. To explore dose–response relationships, correlations were performed between plant growth and end point metrics with soil CNM concentrations, using both two-tailed linear and power regression models. Correlation analyses were conducted both with and without the control data. Statistical analyses were performed using Microsoft Excel 2013, IBM SPSS Statistics 23, and SigmaPlot 12.3.P. vulgaris is characterized by a particular evolutionary history.

Recent analyses based on sequence data presented clear evidence of the Mesoamerican origin of common bean, which was most likely located in México . The expansion of this species to South America resulted in the development of two ecogeographic distinct genetic pools with partial reproductive isolation . After the formation of these genetic pools -between 500,000 and 100,000 years ago – domestication took place, independently in the Mesoamerican and the southern Andean regions of the American continent . Genome analysis of BAT93 and G19833 , P. vulgaris sequenced model genotypes, has initially revealed interesting differences, for example between their genome size and number of annotated genes . The common bean is the most important legume for human consumption. In less favored countries from Latin America and Africa, common bean are staple crops serving as the primary source of protein in the diet. Soil acidity in these tropical regions is a major constraint for crop productivity, usually resulting in a combination of nutrient deficiency and metal toxicity . In acidic soils, aluminum toxicity is the primary factor of growth restriction, resulting in the inhibition of root growth and function, as well as in the increased risk of plants to perish of drought and mineral deficiencies, thus decreasing crop production . High Al levels mainly affect roots causing an arrest of the growth of the principal and lateral roots . In Arabidopsis, the regulation of root growth is modulated by an ABC transporter‐like protein, annotated as ALUMINUM SENSITIVE PROTEIN 3 , which is localized in the tonoplast, suggesting a role in Al vacuolar sequestration . The LOW PHOSPHATE ROOT 1 ferroxidase, an ALS3– downstream protein of the phosphate-deficiency signaling pathway, is involved in root growth inhibition, by modulating iron homeostasis and ROS accumulation in root apical meristem and elongation zone . In root cells, AlT can affect multiple areas, as the plasma membrane, the cell wall and symplastic components .

Common bean is known to be highly sensitive to AlT but this sensitivity is genotype-dependent . In 2010, the evaluation of the root morphological traits related to AlT of 36 P. vulgaris genotypes revealed that Andean genotypes were more resistant to Al than Mesoamerican ones . Mendoza-Soto et al. reported that Mesoamerican common-bean plants subjected to high Al levels for short periods showed decreased root length as well as characteristic symptoms of AlT, such as ROS accumulation, callose deposition, lipoperoxidation and cell death in roots. Along other regulators, plant response to metal toxicity involves also microRNAs as part of the regulatory mechanisms. These molecules are a class of non-coding small RNAs of about 21 nucleotides in length, regulating gene expression at post-transcriptional level, guided by sequence complementarity, inducing cleavage or translational inhibition of the corresponding target transcript . The relevance of miRNA regulation in heavy metal tolerance is well documented; it has been demonstrated that heavy metal-responsive miRNAs show differential expression according to the toxicity level. Target genes of these miRNAs generally encode transcription factors that transcriptionally regulate networks relevant for the response to heavy metals. Additionally these encode transcripts for proteins that participate in metal absorption and transport, protein folding, antioxidant system, phytohormone signaling, or miRNA biogenesis and feedback regulation . High-throughput small RNA sequencing analyses have identified miRNAs that respond to AlT in roots of different plants species, however their function in response to AlT is largely unknown. Some of the target genes cleaved by AlT-responsive miRNAs encode disease resistance proteins, transcription factors or auxin signaling proteins . Our previous research indicated that P. vulgaris is no exception to this phenomenon. We identified common-bean miRNAs that respond to Al, these include conserved miRNAs that are Al-responsive in other plant species -i.e. miR319, miR390, miR393- and also miR1511 . miRNAs from the miR1511 family have been identified in non-legume plants like strawberry and poplar tree ,nft growing system although in the latter its nature as a miRNA has been discussed as it has been considered as part of a retrotransposon . Regarding legumes, miR1511 has been identified in Medicago truncatula and soybean . Also, miR1511 was identified in Mesoamerican common-bean cultivars, being more abundant in flowers and roots . However, this miRNA was not identified when analyzing the Andean G19833 reference genome . Genetic variation in MIR1511 has been reported in a comparative genotyping analysis of different Asian accession of domesticated soybean as well as its wild type progenitor Glycine soja. While sequences of mature miR1511 and miR1511* were found in G. max accessions, the sequences of annual wild G. soja showed insertion/deletion in the stem-loop region of MIR1511 that included complete or partial deletions of mature miR1511 sequence . Updated research indicates that the miR1511 target gene is not conserved in the different plants where it has been identified. In strawberry, the miR1511 targets an LTR retrotransposon gene .

Inconsistencies about the nature of miR1511 target gene also hold for legume species. For instance, different targets have been proposed for soybean ranging from genes coding for proteins involved in the regulation of nitrogen metabolism to proteins relevant in plant cell development . While in other species such as M. truncatula target genes have been searched but have not been identified. The SP1L1 transcript has been proposed as the common-bean miR1511 target , however despite several efforts from our and other groups this prediction could not be experimentally validated. These results suggested a species-specific selection of the corresponding target thus it was essential to experimentally validate the nature and possible function of the miR1511 target gene in common bean. Recent analyses led us to predict an ABC-2-type transporter-related gene, annotated as Aluminum Sensitive Protein 3 , as the target for miR1511. In this work we present its experimental validation. In addition, we genotyped MIR1511 in ecogeographically different common-bean cultivars and investigated the role of miR1511 and its corresponding target in the regulation of plant response to AlT. The comparison of MIR1511 sequence from BAT93 vs. G19833 P. vulgaris reference sequences showed a 58-bp deletion in the G19833 genotype. Such deletion comprised around 57% of pre-miR1511 sequence and included 7-bp and 10-bp of mature and star miR1511, respectively . To explore this phenomenon at a larger scale within the Phaseolus genus, we analyzed Genotyping-By-Sequencing data from 87 genotypes originated from a single genetic population , called non-admixed genotypes. These included genotypes from three Phaseolus species and different populations of wild P. vulgaris: three populations from the Mesoamerican , one from the Andean , and one from the Northern Peru–Ecuador gene pools . All the genotypes belonging to the Andean gene pool and part of the Mesoamerican genotypes displayed a truncated MIR1511, in contrast to the Northern Peru– Ecuador genotypes and the other Phaseolus species that presented a complete version of the MIR1511 in their genome. A population clustering of P. vulgaris genotypes confirmed these results and showed that in the three Mesoamerican populations only a part of the MW1 cluster presented the MIR1511 deletion . Predicted target genes for P. vulgaris miR1511 include SP1L1-like  and isopentyl-diphosphate delta-isomerase , previously reported , and a protein with unknown function and the Aluminum Sensitive Protein 3 , from our recent bio-informatic analysis. From these predicted targets, ALS3 is the only one possibly related to AlT, as reported for Arabidopsis , and showing an adequate binding-site penalty score , thus the 5’RLM-RACE assay was used to experimentally validate the ALS3 mRNA cleavage site. As shown in Figure 3a, a significant number independently cloned transcripts mapped to the predicted site of cleavage, between the nucleotides at positions 457 and 458 of the transcript, which corresponds to position 9 and 10 of the predicted miR1511 binding site, thus confirming a miR1511-induced degradation. The other two degradation events mapped to 7 nucleotides upstream and 17 nucleotides downstream of the miRNA-associated degradation site, suggesting random degradation. An additional action of miR1511 to induce translation inhibition of ALS3 mRNA in common bean, cannot be excluded. miR1511 target genes differ among plant species . In order to evaluate the specificity of the miR1511/ALS3 regulatory node in common bean, we analyzed the miR1511/ALS3 binding site sequence alignment from eight model plant species, including five legumes, which contain a precursor gene of miR1511 in their genome . Because of the deletion in MIR1511 from the G19833 genotype, we used the mature miR1511 and the corresponding ALS3 binding site sequences from the BAT93 Mesoamerican genotype, as representative of P. vulgaris. Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .